Methods and primers for diagnosing idiopathic congenital central hypoventilation syndrome

ABSTRACT

The present invention provides assays and kits for diagnosing idiopathic congenital central hypoventilation syndrome. The present assays and kits focus on the second polyalanine repeat of the PHOX2b gene or gene product, which is normally 20 residues in length. A polyalanine repeat 25 to 33 residues in length is strongly correlated with idiopathic congenital central hypoventilation syndrome.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a divisional of U.S. application Ser. No.10/891,585, filed Jul. 15, 2005, which claims priority to U.S.provisional Application No. 60/488,105, filed Jul. 13, 2003, thecontents of which applications are herein incorporated by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to assays and methods for diagnosing andconfirming the diagnosis of idiopathic congenital centralhypoventilation syndrome.

BACKGROUND OF THE INVENTION

Idiopathic congenital central hypoventilation syndrome (CCHS, also knownas central hypoventilation syndrome, Haddad syndrome and the literarymisnomer “Ondine's curse”; MIM number: 209880, date last edited06-14-04) is an unique disorder of respiratory control [Guilleminault etal., 1982; Haddad et al., 1978; Mellins et al., 1970; Paton et al.,1989; Shannon et al., 1976; Weese-Mayer et al., 1992; Weese-Mayer etal., 1999], occurring in association with Hirschsprung disease (HSCR)[Bower et al., 1980; Commare et al., 1993; Haddad et al., 1978; Hamiltonet al., 1989; Guilleminault et al., 1982; Minutillo et al., 1989; O'Dellet al., 1987; Stem et al., 1980; Verloes et al., 1993; Weese-Mayer etal., 1992], tumors of neural crest origin [Bower et al., 1980; Commareet al., 1993; Haddad et al., 1978; Swaminathan et al., 1989; Weese-Mayeret al., 1992] (neuroblastoma, ganglioneuroblastoma, ganglioneuroma), andsymptoms of diffuse autonomic nervous system dysfunction (ANSD)[Weese-Mayer et al., 2001] including decreased heart rate variability[Ogawa et al., 1993; Silvestri et al., 2000; Woo et al., 1992], anattenuated heart rate response to exercise [Silvestri et al., 1995],severe constipation [Weese-Mayer et al., 1992], esophagealdysmotility/dysphagia [Faure et al., 2002], decreased perception ofdiscomfort, pupillary abnormalities [Goldberg et al., 1996; Weese-Mayeret al., 1992], decreased perception of anxiety [Pine et al., 1994],sporadic profuse sweating, and decreased basal body temperature amongothers. Subsequently, symptoms of ANSD have been identified in nuclearfamily members of the probands with CCHS, though the relatives of theCCHS cases tend to manifest a milder spectrum of ANSD, with fewersymptoms and/or fewer systems than the cases [Weese-Mayer et al., 2001].

CCHS is thought to be genetic in origin based upon familial recurrencedata, and genetic segregation analyses. Familial recurrence data includeone report each of affected monozygotic female twins [Khalifa et al.,1988], sisters [Haddad et al., 1978], male-female sibs [Weese-Mayer etal., 1993], and male-female half sibs [Hamilton et al., 1989] with CCHS.More recently, a total of five women diagnosed with CCHS in their ownchildhoods have given birth to infants including two with definite CCHS,one with likely CCHS confounded by severe immaturity andbronchopulmonary dysplasia, and one with later onset CCHS [Silvestri etal., 2002; McQuitty personal communication; Sritippayawan et al., 2002].A recent report of a child with CCHS born to a woman who hadneuroblastoma as an infant [Devriendt et al., 2000] provides additionalevidence for a transmitted genetic component in the phenotypic spectrumof ANSD and CCHS. Further, ANSD has been studied in a case-controlfamily design, including families ascertained through a CCHS-affectedchild and families of matched controls. Segregation analysis of aquantitative ANSD trait in such families found that the best-fittingmodel for ANSD was codominant Mendelian inheritance of a major gene[Marazita et al., 2001]. These results support the prior hypothesis thatCCHS is the most severe manifestation of a general ANS dysfunction.[Weese-Mayer et al., 1993].

Pursuit of the genetic basis for CCHS by molecular genetic analysis hasbeen limited due to the rarity of the disease (likely fewer than 400cases worldwide). To date, most studies have also been limited to thestudy of genes known to be related to Hirschsprung disease. Thus far,three discrete variants which alter a single amino acid in RET, a cellsurface tyrosine kinase receptor, have been reported in three unrelatedCCHS patients [Amiel et al., 1998; Sakai et al., 1998; 2001]. Thesealterations were also present in one or both parents for two cases. Amutation in glial cell line-derived neurotrophic factor (GDNF) wasreported in another patient and his unaffected mother [Amiel et al.,1998]; a mutation in endothelin-3 was reported in a fifth patient [Bolket al., 1996]; and a mutation in brain-derived neurotrophic factor[Weese-Mayer et al., 2002] was reported in a sixth patient with symptomsof ANSD in his father. Three other reports indicate an absence of RETmutations [Bolk et al., 1996] and RNX mutations [Amiel et al., 2002;Matera et al., 2002].

Unfortunately, the success of studies that have focused on genes knownto be related to Hirschsprung disease has been limited. Although, therehas been a report of heterozygous expansion mutations in a polyalaninetract within PHOX2b in 18 of 29 children with CCHS in France [Amiel etal., 2003]. The expansion mutations were determined by direct sequencingof the PHOX2b gene. As is well known in the art, direct sequencing is anarduous and time intensive task.

Accordingly there remains a need for a simple, efficient assay forhelping to diagnose CCHS.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a method of diagnosing orconfirming idiopathic congenital central hypoventilation syndrome (CCHS)in a subject. The method can include assaying for a polyalanine repeatexpansion mutation in the second polyalanine repeat of the PHOX2b geneor gene product of a subject. In other words, the method provides fordetermining whether the subject has a polyalanine repeat expansionmutation in the second polyalanine repeat of the PHOX2b gene or geneproduct. In some embodiments of assays of the invention, a polyalaninerepeat 25 to 33 alanine residues in length indicates that the subjecthas, had or is at risk for CCHS. In some embodiments of the presentmethods, the assay is not performed by direct sequencing or by singlestrand conformation polymorphism analysis. Generally, the methodidentifies about 65, 70, 75, 80, 85, 90, 95, 97 percent or more ofsubjects having CCHS.

Another embodiment provides a method of determining whether an offspringof a subject is at risk for idiopathic congenital centralhypoventilation syndrome. This method can involve determining whetherthe subject has a polyalanine repeat expansion mutation in the secondpolyalanine repeat of a PHOX2b gene or gene product, such as through anassay. In these methods, the presence of a polyalanine repeat 25 to 33alanine residues in length indicates that the offspring of the subjectis at risk for CCHS.

In the above embodiments, the methods can further include obtaining asample from the subject that includes a nucleic acid containing thesecond polyalanine repeat located within exon 3 of the PHOX2b gene. Inthese methods, the sample can include blood, white blood cells,epithelial cells, skin, hair, fibroblasts, a tissue from an organ,amniocytes, chorionic villi, embryonic cells, polar bodies, sperm andcombinations thereof as desired. These and other methods can furtherinvolve amplifying the nucleic acid containing the polyalanine repeat ofthe PHOX2b.

In any of the above methods, the assay or determining can includequantifying the length of the polyalanine repeat of the PHOX2b gene orgene product of the subject. In yet other embodiments, the PHOX2b genecan be directly assayed to determine whether it codes for a polyalaninemutation. In still other embodiments, the methods can be used todetermine whether the gene product, e.g. the PHOX2b RNA or protein,codes for or has a polyalanine mutation.

In some of the above methods, the PHOX2b gene or gene product can beseparated and the size of the PHOX2b gene or gene product can bedetermined by comparison against a known standard. Examples ofseparation techniques include chromatography techniques. Anychromatographic method that separates the gene or gene product fromother components in the sample can be used in the present invention.Non-limiting examples of chromatographic methods include HPLC, dHPLC andthe like. The separation can also be performed by electrophoresis.

The present methods can further include confirming whether the subjecthas the polyalanine mutation in the polyalanine repeat of the PHOX2bgene or gene product by sequencing the PHOX2b gene or gene product,including the polyalanine repeat region.

The present methods can be used to identify PHOX2b mutations indicativeof CCHS in subjects or their offspring that have been diagnosed, atleast presumptively, with sudden infant death syndrome. Some of thesubjects or their offspring diagnosed by the present methods can haveHirschprung disease, alveolar hypoventilation, a tumor of neural crestorigin and combinations of these diseases and disorders. In someinstances, there can be a correlation between the polyalanine repeat ofthe PHOX2b gene or gene product and the severity of the symptoms ofautonomic nervous system dysregulation characteristic of CCHS.Accordingly, the present methods can be useful for identifyingtherapeutic regimens for subjects having CCHS. The present methods canfurther include determining whether a parent of the subject has apolyalanine mutation in a polyalanine repeat of a PHOX2b gene or geneproduct, such as by an assay. In any of the above methods, the subjectcan be an embryo, fetus, child, juvenile or adult. Although the methodsand assays of the present invention have special significance in thediagnosis and confirmation of fetal, infant and juvenile CCHS, theskilled artisan will understand that the methods and assays may be usedto diagnose or confirm CCHS in a subject of any age. For example, themethods and assays of the present invention may be used to determine ifan older infant, child, or adult, including someone over the age of oneyear as well as someone over the age of 20 years, has late onset centralhypoventilation syndrome. Generally, if the term proband is used, itdenotes the family member through whom a family's medical history comesto light. In some of the present methods, the subject is human.

The present invention also provides kits for performing the presentmethods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Polyalanine expansion mutation in individuals and families withCCHS: (A) Polyacrylamide electrophoresis of products from CCHS subjects(lanes 1-9) and controls (lanes 10-12). Genotypes in terms ofpolyalanine repeat numbers are as follows: 20/27 (lanes 1, 2, 9), 20/26(lanes 3, 7), 20/33 (lane 4), 20/25 (lanes 5, 6), 20/28 (lane 8), 20/20(lanes 10-12). (B) CCHS mutation in family segregating deletion variant.The proband (lane 1) has alleles of 15 and 26 repeats while the father(lane 3) carries the deletion variant with 15 repeats in addition to hisnormal allele of 20 repeats. The mother (lane 2) and two siblings (lanes4, 5) have only the normal 20 repeat allele. (C) CCHS proband and motherwho is mosaic for expansion mutation. The proband (lane 1) has genotype20/26 while the mother (lane 4) also has this genotype but the bandintensity of the mutated 26 repeat allele is much lighter than that ofthe normal allele. The father (lane 2) and both maternal grandparents(lanes 3, 5) have only the normal 20 repeat allele. (D) Rapid screeningfor the expansion mutation by agarose electrophoresis which identifieseven the smallest expansions. Molecular weight markers are in lane 1.Control with 20/20 genotype is in lane 2. Probands with CCHS expansionmutation have genotypes 20/25 (lane 3), 15/26 (lane 4) 20/27 (lane 5),and 20/33 (lane 6).

FIG. 2. Allele frequency distribution of PHOX2b polyalanine repeatexpansion mutations in 65 CCHS probands and 3 infants with CCHS born toCCHS probands. Among the 13 cases with Hirschsprung disease and/or atumor of neural crest origin as well as the polyalanine repeat expansionmutation, 5 cases (38%) have the longest polyalanine repeats (8-13). The2 children with ganglioneuromas have the 13 repeat; 3 children withHirschsprung disease have 13, 10, and 8 repeats.

FIG. 3. PHOX2b polyalanine repeat expansion mutation in CCHS probandsand their offspring: (A) mother (lane 1) and child (lane 2) both withexpansion mutation (genotype 20/25) and CCHS; (B) mother (lane 2) withexpansion mutation (20/25) and CCHS and child (lane 1) with normalgenotype (20/20) and no symptoms of CCHS.

FIG. 4. Number of ANSD symptoms in CCHS probands vs. PHOX2b polyalaninerepeat expansion mutation length. Many subjects had identical numbersfor ANSD symptoms and polyalanine repeats, therefore the figure givesthe illusion of fewer data points than expected for the 65 CCHS caseswith the PHOX2b mutation.

DETAILED DESCRIPTION

The present invention provides a method or assay for diagnosing orconfirming the diagnosis of CCHS in a subject. The present methods focuson the second polyalanine repeat of the paired-like homeobox 2b(PHOX2b), protein and the nucleic acids, both DNA and RNA, that code forthe protein. PHOX2b has also been referred to as NBPhox and PMX2B. Someassays determine the size of the PHOX2b exon 3 gene sequence coding forthe polyalanine repeat in order to identify individuals affected withsymptoms of CCHS or at risk of passing a CCHS mutation to theiroffspring. Other assays detect the downstream gene products, such as RNAor proteins, of the PHOX2b gene to determine the number of polyalaninerepeats contained or coded for by the gene product.

PHOX2b maps to chromosome 4p12 and encodes a highly conserved homeoboxdomain transcription factor (314 amino acids in length), with two shortand stable polyalanine repeats of 9 and 20 residues. [Amiel et al.,2003] PHOX2b has an early embryologic action on pan-neuronaldifferentiation including upregulation of proneural gene expression andMASH1 expression [Dubreuil et al., 2002]. PHOX2b also has a separaterole by a different pathway wherein it represses expression ofinhibitors of neurogenesis [Dubreuil et al., 2002]. Further, PHOX2b actsas transcriptional activator in promotion of generic neuronaldifferentiation and expression of motoneural differentiation [Dubreuilet al., 2002]. Finally, PHOX2b is required to express dopamine betahydroxylase [Lo et al., 1999], RET and MASH1 as well as tyrosinehydroxylase, thereby indicating regulation of PHOX2b over thenoradrenergic phenotype in vertebrates [Pattyn et al., 1999]. This roleof PHOX2b early in the embryologic origin of the ANS with a role indetermining the fate of early neuronal cells and a role in disinhibitionof neuron differentiation might account for the seeming imbalance in thesympathetic and parasympathetic nervous system in children with CCHS.

Sequences reported for the PHOX2b gene with or without the promoterregion and 5′ untranslated region are disclosed as accession numbersAF117979, AB015671, BC017199, NM_(—)008888.1 and D82344 available in theNCBI database at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi.

The wild type PHOX2b cDNA has the sequence (Accession No. AF117979):

SEQ ID NO: 1 ttaaatttta attagagatg caggatcaat gatagggagt tggacagttcagttccccag tgccagccca atagacggat gagttatttt catgtaaaaa gcgccagcaataagaccaac cgtttgttat tgtcccaagt ggaaagagcc aagtttatta tgaggactatatggttttag agacttcaga caaggcatnt cataggaggc tttttcataa aactaggntctgctggtagt aaggaggcca gtttggaggc aggcgttgag ctgtgcacat ctccccactccagccacctt ctccatatcc atcttttatt tcatttttcc acttggctga gccatccagaaccttttcaa tgtataaaat ggaatattct tacctcaatt cctctgccta cgagtcctgtatggctggga tggacacctc gagcctggct tcagcctatg ctgacttcag ttcctgcagccaggccagtg gcttccagta taacccgata aggaccactt ttggggccac gtccggctgcccttccctca cgccgggatc ctgcagcctg ggcaccctca gggaccacca gagcagtccgtacgccgcag gtaaggacct tcagctttct cagcggagga agccgccttt ccgcccgtatataggaagcc ttgattgcat ttgaaaatgg aaatgtgttt agtatttacc aaacgaaatttgcttacaca aatgaaagaa tttatcacgt tagaagcgat tgcagggagg ggtaattcacttacagggtt acactatcct agtcacaccc gaaccgccaa caaaattatc ttaagctgccaaaatgatag gcataattta tttactttgc gatgagacgt aaagcttaga aaataattaaataacaaaga gtaaagctca ttactggcag tgtctctttt tttaagaacc gacagcggctcacacctctt tggctggtca tttttatgat tatttcttta atttattatt atttttttgcagctctttcc cccaactttt gagccgggtc aactttctga gaattgaaaa gttcccaaagtgggactgtt tggtaacttc tttcctggct ccctgatatt ccgactgatg tttttggatttttttcctct ctggtttttt cctgctgaaa gcactatctc aagtccgtca catcgcgctgtttcaatcca cccaaaggcg cttgtgccag aaaggactcc gccaagcccg aagtttgagcccaggtttcc gcagataaca aatttcctcg gtttcttccc gcagcttctc tcggcaactctctcgcgcgg gtgtaggtag cggctgccgt atgacctgac cttggagtcc tcacattctagctccacggc cggcgagctg ccggctgatt tgctcacttt ctgtctcctc tgtcatactctagttcctta caaactcttc acggaccacg gcggcctcaa cgagaagcgc aagcagcggcgcatccgcac cactttcacc agtgcccagc tcaaagagct ggaaagggtc ttcgcggagactcactaccc cgacatctac actcgggagg agctggccct gaagatcgac ctcacagaggcgcgagtcca ggtacgcgcg cctggaaacc gaccccgctc cgccgcactg gtccggggaggtgtggggtg aggggcggct ggtaaattcg aagtcctgga gcctcgagtg agaaggacctagggccccat ggccgatcag aaatactgga tttggtgtgg ctgtgcgttc gagagaggcttagagcgcac gctcttggca ttttatttac agttgcgaag tgtttcccac ccgagcagagacatgggggg ccttgggacg tggatgagcg atgcaatttc ggggacagga agtgcctgtggtggaaggtg tgcagacttt gctcccgtat tataagtttt tccttctccc ctcccgccccccaaaaaaat gcctcctaac tcaagtgctt ttaacctggc cccatggcat ataggttcattttcccggaa actgtgactt gcatcagatt tgcaaagggt ctgtgacttc atgaaggtcaagaaccatga cttactccaa cctgttaaac acaggtgcgc tcacgagttg gccacagcgcctctctgggt gagcccccga ccgagaagcg gtgcgcacca tcgcacgctc ttccaggctcaaaggccggg gatgggcagc ggagcaaacc cagaggatcc cttttccttc taccaattagagtttaactt tagaacttag gcttaggggt gaatggcgag ctcggggctt gctcaagaagccgacttgaa cagaggccca ccaaaataag gccttccctt ttcgggtctt tctgggacctgcggcttttt aaactctgcc gcaagccttc atgtccctgg cgtgctcact ccccctaagaaagtttctcc gaaaatgcac agcaataaga agcggtagac ttggtggatg tgcgcgcgggggtgatcaca gcgcatgggg aggagggtgt taaaacaagc cgaagtagaa cttgggccaccctaaccggt gcttttcttt cccattttct tctttctccc cctgcttcac cgtctctccttccgtcttgg gccaggtgtg gttccagaac cgccgcgcca agtttcgcaa gcaggagcgcgcagcggcag ccgcagcggc cgcggccaag aacggctcct cgggcaaaaa gtctgactcttccagggacg acgagagcaa agaggccaag agcactgacc cggacagcac tgggggcccaggtcccaatc ccaaccccac ccccagctgc ggggcgaatg gaggcggcgg cggcgggcccagcccggctg gagctccggg ggcggcgggg cccgggggcc cgggaggcga acccggcaagggcggcgcag cagcagcggc ggcggccgcg gcagcggcgg cggcggcagc ggcagcggcggcagctggag gcctggctgc ggctgggggc cctggacaag gctgggctcc cggccccggccccatcacct ccatcccgga ttcgcttggg ggtcccttcg gcagcgtcct atcttcgctccaaagaccca acggtgccaa agccgcctta gtgaagagca gtatgttctg atctggaatcctgcggcggc ggcggcggcg gcgacagcgg gcgagccagg gcccgggcgg gcgagtgggcgagcgggtag gcccaaggct attgtcgtcg ctgctgccat ggctttttca ttgagggcctaaagtaatcg cgctaagaat aaagggaaaa cggcgtcgcc ctcatttcaa ccccactcctacccccttcc tcaaccccca aacaaaacaa acaaacttcc ctggcttcgc acctgcctggggcctcgcag cggggccagg gctccgcctg ctgatcgggg gttgtgagca gcgcggcctggacgcggggc actctcaggg ggctgtgtct gcgtgtcagt ttgtgtctgt ctcggggaatgtgtgtctgt ggcccaagca ggtgacagga agagatgggg ggcctcaacc aacttagtgacttgtttaga aaaaaaagac aaaaaagtaa aaataaaaac aaaaaagttg gaaggcagaaaccattaaaa aacaaaaagc caacaaccca gaaaggttta aaaaacataa ggaaaaaaaagacaaattaa aggaggggct aggggagaag ctgcagctgg agctgaaggc tcgatcttgtgaacccctaa atccgctccc tcctaacagc acggattctc ttggggctct tcttcagggaagagtaggga cgccgttcca gccccccttc ctatcgtgtc cttgggttcg ggtcactgcggcgacgactt gctcagactg tcccggcggc cggagtgact ttctcgcacc cccttgcctgtcccacctcg ctgaacacca tcccgccatt agcgcatcgg aaccccacac agttgcaactcccaaccccg aatctttgca gccgttcggc cctgaaagat gccctatcca tgagatgccttttcatctgc aaactctgca aaatgtgtct catgtttcgc aactcttttt ttccccctcgctcccgccta ccccgtcggc attttcttct tccaccagct tttactgaac tttttggcactgctttggat tggggtcaat tgcagtccac gtaactggct gcagagaaat ctaccgagcaaggaaaaggc acacacacac gtttgcaggg gtgtctcggt ttgcatttct gttggaatgatccgaactgg actcacatcc tgtatggtgg atggactgta tattgagggt tccattcttcgcgcagttta gacatctctg ttttgattct ttgttgttgt ttttatttta aaaggcacaaactctagata ttagttgaat gttgaggctt taactttttc ggtgtctttc tacaactgtgttctgtgact caattgtatc gtgttaatat cagtgcagac tgtctcctct acgtgaccgtataatgtttt tctcgtcttg tagtctctat ggcgtgtctt tatggtgtaa taaggttctcacggggtcaa tcttttgtgt ttagagaggc cacggttcag acaatggtat atatttttgttatcaggtgc atgtctgtct gatttctttt tttttcctgt tggactatgt ttgtgaacataattgtcata agttatgttt cagatttttg aatttattta tatgtgttat aatgaatgcttctatttaaa agggaaatat ttctacatgt gcttatagtt ttccaagagt gtaccattaacttgattgtt gataataaaa accaaaagca agtct.Adachi et al., DNA Cell Biol. 19 (9), 539-554 (2000); Yokoyama et al.,Genomics 59 (1), 40-50 (1999), Pattyn et al., Nature 399 (6734), 366-370(1999); Pattyn et al., Development 124 (20), 4065-4075 (1997); andYokoyama et al., DNA Res. 3 (5), 311-320 (1996).

The wild-type protein encoded by the PHOX2b gene in humans has thesequence (Accession No. AAD26698; corresponding amino acid sequence toAccession No. AF117979):

SEQ ID NO: 2 mykmeysyln ssayescmag mdtsslasay adfsscsqas gfqynpirttfgatsgcpsl tpgscslgtl rdhqsspyaa vpyklftdhg glnekrkqrr irttftsaqlkelervfaet hypdiytree lalkidltea rvqvwfqnrr akfrkqeraa aaaaaaakngssgkksdssr ddeskeakst dpdstggpgp npnptpscga nggggggpsp agapgaagpggpggepgkgg aaaaaaaaaa aaaaaaaaaa gglaaaggpg qgwapgpgpi tsipdslggpfgsvlsslqr pngakaalvk ssmf.Adachi et al., DNA Cell Biol. 19 (9), 539-554 (2000); Yokoyama. et al.,Genomics 59 (1), 40-50 (1999), Pattyn et al., Nature 399 (6734), 366-370(1999); Pattyn et al., Development 124 (20), 4065-4075 (1997); andYokoyama et al., DNA Res. 3 (5), 311-320 (1996).

The wild-type protein encoded by the PHOX2b gene in mus musculus has thesequence (Accession No.: NP_(—)032914; corresponding amino acid sequenceto Accession No. NM_(—)008888.1)

SEQ ID NO: 3 mykmeysyln ssayescmag mdtsslasay adfsscsqas gfqynpirttfgatsgcpsl tpgscslgtl rdhqsspyaa vpyklftdhg glnekrkqrr irttftsaqlkelervfaet hypdiytree lalkidltea rvqvwfqnrr akfrkqeraa aaaaaaakngssgkksdssr ddeskeakst dpdstggpgp npnptpscga nggggggpsp agapgaagpggpggepgkgg aaaaaaaaaa aaaaaaaaaa gglaaaggpg qgwapgpgpi tsipdslggpfasvlsslqr pngakaalvk ssmf.

Yokoyama et al., Genomics 59 (1), 40-50 (1999), and Pattyn et al.,Development 124 (20), 4065-4075 (1997).

The present assays of the PHOX2b polyalanine repeat mutation canrepresent a highly sensitive and specific technique for confirming thediagnosis of CCHS. Identification of the CCHS mutation can further leadto clarification of the phenotype, allow for prenatal diagnosis forparents of CCHS probands and adults at risk for having children infuture pregnancies with CCHS, and potentially direct interventionstrategies for the treatment of CCHS.

In a specific embodiment, the present methods involve an amplificationassay for diagnosis of Idiopathic Congenital Central HypoventilationSyndrome (CCHS) by detection of polyalanine repeat expansion mutationsin the PHOX2b gene. In this embodiment, a nucleic acid amplification,such as a polymerase chain reaction-based (PCR) assay is used to detectand/or determine the size of polyalanine expansion mutations in thePHOX2b gene. The amplification reaction contains nucleic acid, such asDNA or RNA, from the individual who is to be tested, and all thenecessary components for amplifying the relevant portion of the nucleicacid, such as oligonucleotide primers specific for nucleic acidsequences flanking the repeat-coding area of the PHOX2b gene,nucleotides, a polymerase, salts, and buffer. The reaction mixtures aresubjected to amplification, typically in a thermal cycler, and theamplification products are separated from other components of thereaction, such as by electrophoresis on a gel, so that the size of theproducts, which reflect the size of the gene sequence, or the geneproduct sequence, of the individual to be tested, can be determined. Thenumber of repeats is determined from comparison of the amplificationproducts with size standard, such as nucleic acids that have knownsizes, including those with known repeat sizes. The assay has been usedto detect an expansion mutation, or larger number of repeats, invirtually all individuals with CCHS.

In some embodiments, 7-deaza-dGTP is used in the nucleotide mix in placeof some, e.g. up to 70% or more, or all dGTP to improve nucleic acidamplification and/or isolation.

In one aspect, nucleic acid amplification may be used to isolate fromgenomic nucleic acid a substantially pure DNA or RNA (i.e., a DNA or RNAsubstantially free of contaminating nucleic acids) encoding the entirePHOX2b gene or a part thereof. In some embodiments, such a DNA or RNA isat least 95% pure, more preferably at least 99% pure. In certainembodiments, any of the oligonucleotide sequences, degenerate orotherwise, that correspond to peptide sequences of PHOX2b disclosedherein can be used as primers.

The present assays can also use various probes and probing strategiesthat are specific for the target PHOX2b nucleic acid gene or geneproduct sequences to determine the number of polyalanine repeats thenucleic acids code for. In this embodiment, the assay can involve addinga probe or number of probes, such as nucleic acids specific for a givennumber of polyalanine repeats to a sample containing the PHOX2b sequenceof interest and determining whether the probe binds to the PHOX2bnucleic acid. In one embodiment, one probe that is specific for thenaturally occurring 20 alanine repeat can be used to determine whetherthe nucleic acid sample contains only the naturally occurring 20polyalanine repeat or whether the nucleic acid sample also containsnucleic acids with more or less polyalanine repeats than the 20polyalanine repeat. One or more probes that are specific for differentpolyalanine repeat lengths, for example 25 to 33 repeats, canadditionally or alternatively be used in the assay. In the presentmethods, the sequence or identity of the probe(s) is (are) notparticularly limited as long as the probe(s) can discriminate betweenthe naturally occurring 20 polyalanine repeat and the polyalaninerepeats that are indicative of CCHS. One skilled in the art can readilyproduce such probes based on the identified nucleic acid and/or proteinsequence of PHOX2b. In some probe embodiments, the probes will not onlyhave sequences complementary to those found in the polyalanine repeatcoding sequence they will also have sequences, for example from three to20 bases in length, that correspond to one or both of the sequencesadjoining the polyalanine repeat coding sequence. In some embodiments,one or both of the probe(s) and target PHOX2b nucleic acid will belabeled with a detection moiety.

Specificity of the probe-target binding can be determined through anymeans known in the art, such as by determining the Tm of the nucleicacid complex. In some of these assays, the results can be comparedagainst one or more control samples that contain nucleic acid with thenaturally occurring 20 polyalanine repeat and/or mutants having a knownnumber of polyalanine repeats.

Other embodiments of the present methods involve directly assaying apeptide or protein produced from the PHOX2b gene to determine the numberof polyalanine repeats present in the protein. The size of the proteinand number of polyalanine repeats can be determined in any suitablemanner, such as by isolating the protein and/or separating the protein.As above, the size of the protein can be compared against controlproteins that have known, defined sequences and sizes. The peptides orproteins may also be sequenced. The present invention also contemplatesthe use of antibodies, or fragments thereof, that are specific forPHOX2b proteins that have different polyalanine repeat lengths.Production of antibodies and antibody fragments specific for a targetprotein are well known in the art [Pattyn et al., 1997] and will not bediscussed herein in detail.

Sample nucleic acid or protein to be analyzed by any known diagnosticand prognostic methods can be obtained from any cell type or tissue of asubject. For example, a subject's bodily fluid (e.g. blood) or cells canbe obtained by known techniques (e.g. venipuncture, biopsy or the like).Alternatively, nucleic acid tests can be performed on dry samples (e.g.hair or skin). Fetal nucleic acid samples can be obtained from maternalblood as described in International Patent Application No. WO91/07660(Bianchi). Alternatively, amniocytes or chorionic villi may be obtainedfor performing prenatal testing. The genomic DNA used for the diagnosismay be obtained from body cells, such as those present in peripheralblood, urine, saliva, buccal mucosa, surgical specimen, and/or autopsyspecimens. The DNA may be used directly or may be amplifiedenzymatically in vitro through use of PCR (Saiki et al. Science239:487-491 (1988)) or other in vitro amplification methods such as theligase chain reaction (LCR) (Wu and Wallace Genomics 4:560-569 (1989)),strand displacement amplification (SDA) (Walker et al. Proc. Natl. Acad.Sci. U.S.A. 89:392-396 (1992)), and/or self-sustained sequencereplication (3SR) (Fahy et al. PCR Methods Appl. 1:25-33 (1992)), priorto mutation analysis. The methodology for preparing nucleic acids in aform that is suitable for mutation detection is well known in the art.

The detection of mutations in specific nucleic acid sequences can beaccomplished by a variety of methods including, but not limited to,restriction-fragment-length-polymorphism (RFLP) detection based onallele-specific restriction-endonuclease cleavage (Kan and Dozy Lancetii:910-912 (1978)), hybridization with allele-specific oligonucleotideprobes (Wallace et al. Nucl Acids Res 6:3543-3557 (1978)), includingimmobilized oligonucleotides (Saiki et al. Proc. Natl. Acad. Sci. U.S.A.86:6230-6234 (1989)) or oligonucleotide arrays (Maskos and Southern NuclAcids Res 21:2269-2270 (1993)), allele-specific PCR (Newton et al. NuclAcids Res 17:2503-2516 (1989)), mismatch-repair detection (MRD) (Fahamand Cox Genome Res 5:474-482 (1995)), binding of MutS protein (Wagner etal. Nucl Acids Res 23:3944-3948 (1995), denaturing-gradient gelelectrophoresis (DGGE) (Fisher and Lerman et al. Proc. Natl. Acad. Sci.U.S.A. 80:1579-1583 (1983)), RNAase cleavage at mismatched base-pairs(Myers et al. Science 230:1242 (1985)), chemical (Cotton et al. Proc.Natl. Acad. Sci. U.S.A. 85:4397-4401 (1988)) or enzymatic (Youil et al.Proc. Natl. Acad. Sci. U.S.A. 92:87-91 (1995)) cleavage of heteroduplexDNA, methods based on allele specific primer extension (Syvanen et al.Genomics 8:684-692 (1990)), genetic bit analysis (GBA) (Nikiforov et al.Nucl Acids Res 22:4167-4175 (1994)), the oligonucleotide-ligation assay(OLA) (Landegren et al. Science 241:1077 (1988)), the allele-specificligation chain reaction (LCR) (Barrany Proc. Natl. Acad. Sci. U.S.A.88:189-193 (1991)), gap-LCR (Abravaya et al. Nucl Acids Res 23:675-682(1995)), and radioactive and/or fluorescent nucleic acid sequencing orlabeling using standard procedures well known in the art.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of subject tissue obtained from biopsiesor resections, such that no nucleic acid or protein purification isnecessary. Nucleic acid reagents may be used as probes and/or primersfor such in situ procedures (see, for example, Nuovo, G. J., 1992, PCRin situ hybridization: protocols and applications, Raven Press, N.Y.).

As used herein, a gene product or product of a gene refers to a moleculethat is produced based on the sequence of the gene. Examples of geneproducts include RNA, such as mRNA, and proteins.

PHOX2b analyses in the present examples were also extended to parentsand other family members of CCHS cases, and parent/infant pairs in whichthe mother had CCHS. The PHOX2b assays in the examples were specificallyundertaken to develop a simple and accurate assay for sizing of therepeat sequence associated with the polyalanine tract expansion, andclarify inheritance patterns for CCHS mutations in families.

In the methods and assays embodied in the examples, 65/67 CCHS probands(97%) were found to be heterozygous for the exon 3 polyalanine expansionmutation of PHOX2b. Further, there was an association between repeatmutation length and severity of the CCHS/ANSD phenotype. Of the twoprobands who did not carry the expansion mutation, one had a nonsensemutation in exon 3 that truncated the protein and the other had nomutation in PHOX2b but had a previously reported endothelin 3 (EDN3)frameshift point mutation. The polyalanine expansion mutation was notfound in any of 67 matched controls. Of clinically unaffected (no CCHS)parents from 54 available, families (43 parent pairs and 11 singleparents) whose child carried the PHOX2b mutation in the example, fourparents from the 43 parent pairs demonstrated mosaicism for an expansionmutation identical to that seen in the CCHS cases, suggesting that notall mutations in affected probands with unaffected parents are de novo.Four women with CCHS who were heterozygous for the PHOX2b mutation, eachwith one child, were also studied. Three of the four children were alsoaffected and had the same mutation, demonstrating autosomal dominantinheritance of the mutation.

Based on certain embodiments of the present assays, demonstrated by theexamples, PHOX2b was found to have a statistically significantassociation with CCHS. It appears that children with CCHS areheterozygous for a polyalanine expansion mutation in PHOX2b. The resultsset forth in the examples suggest that heterozygosity for a mutation inPHOX2b is sufficient to produce CCHS. The results of the methods andassays of the examples demonstrate the finding of unique PHOX2bmutations in some patients with the triad of Hirschsprung disease,alveolar hypoventilation, and neuroblastoma. Although the mutationsidentified were not identical, it is of great interest nonetheless, withthe only other unique mutation found in one French study patient withHirschsprung disease and alveolar hypoventilation. These observationsindicate the importance of PHOX2b gene sequencing in patients believedto carry the CCHS phenotype but who do not have the polyalanine repeatmutation.

Surprisingly and unexpectedly, as shown by the results of the examples,embodiments of the present methods and assay provided a much highersuccess rate in identifying patients with CCHS than previous references.As can be seen from the examples, the present assay identified thePHOX2b polyalanine expansion mutation in 97% of cases and any PHOX2bmutation in 98.5% of CCHS cases, whereas prior experiments onlyidentified 62% and 69% of patients with the PHOX2b polyalanine expansionmutation or any PHOX2b mutation, respectively. Accordingly, the presentmethods can specifically exclude techniques such as direct sequencing ofnucleic acids and/or single strand conformation polymorphism analysis.Additionally or alternatively, the present invention also provides anassay that identifies about 65, 70, 75, 80, 85, 90, 95, 97 percent ormore of subjects that have, or are at risk for, CCHS.

Embodiments of the invention allow determination of an associationbetween PHOX2b polyalanine repeat mutation length and severity, asmeasured by numbers of ANSD symptoms and daily duration of requiredventilatory support. This determination was surprising and unexpected.

Additionally, using individual embodiments and methods of the presentinvention, the present inventors have discovered that offspring of asubject are at higher risk for CCHS when the subject has a polyalanineexpansion mutation, for example where the subject is a somatic mosaicfor the polyalanine expansion mutation. This is in contrast to previousstudies that did not identify PHOX2b polyalanine repeat mutations inparents of CCHS probands, but instead suggested that the mutation arisesas a de novo event in subjects with CCHS. As demonstrated in theexamples, the present methods and assays did find a polyalanine repeatmutation, apparently present as a somatic mosaic, in 4 asymptomatic (forCCHS) parents, suggesting that in a small (˜10%) percent of familieswith a child with CCHS, there is a risk of recurrence in a second child.The finding of the mosaic carrier parents is consistent with priorreports of sibships with CCHS [Haddad et al., 1973; Hamilton et al.,1989; Khalifa et al., 1988; Weese-Mayer et al., 1993]. Parental mutationscreening is therefore important after identification of a child withCCHS and a PHOX2b mutation, in order to provide appropriate counselingto the family regarding recurrence risk. Given that somatic mosaicismexists in some families, it is possible that germline mosaicism alsoexists; after birth of a child with CCHS, prenatal testing should beperformed to predict the affection status of subsequent pregnancies.Only the embodiments of the present invention are known to be sensitiveenough to provide this screening.

Transmission of CCHS from parents with CCHS to their children wasaddressed through the study of four mother-child pairs in which themother had CCHS and the PHOX2b polyalanine expansion mutation. In thesecases, the mutation and the disease were transmitted in an autosomaldominant fashion: inheritance of one copy of the expanded alleleresulted in disease, while inheritance of the normal 20 repeat allele(from both parents) did not result in disease. One of the affectedchildren was initially considered to be asymptomatic, but prior todetermining that he was carrying the PHOX2b mutation he becameventilator-dependent during sleep after a viral illness. Thisobservation suggests that presence of the PHOX2b expansion mutation asdemonstrated by using the methods and assays of the invention is highlypredictive of disease, even in an infant not initially showing signs ofCCHS. As shown in the embodiments of the examples, in all three cases inwhich the expansion mutation was transmitted from mother to child, therewas no change in the number of repeats and mother and child carried thesame alleles. Likewise, in all four cases where a CCHS parent was amosaic, the expansion mutation was transmitted parent to child with nochange in the number of repeats and the mosaic CCHS parent and CCHSproband child carried the same alleles. The results from this groupsuggest that the polyalanine expansion in PHOX2b is meiotically stable.In combination with the notable absence of the 1-4 repeat expansions incontrol and CCHS groups, these results support that the mutation islikely to occur through mispairing during replication followed byunequal crossing over rather than through a strand slippage mechanism.However, the skilled artisan will understand that embodiments of theinvention may work through separate mechanisms.

Extensive studies by Pattyn et al. [1997, 1999] indicate an earlyexpression pattern of PHOX2b in rhombencephalon, suggesting a link toearly patterning events with later neurogenesis in the hindbrain. In themouse, PHOX2b is expressed in the neonatal CNS, specifically in the areapostrema, nucleus tractus solitarius, dorsal motor nucleus of the vagus,nucleus ambiguus, ventral surface of medulla, locus coeruleus (untilembryonic day 11.5), and the IIIrd (oculomoter), IVth (trochlear), VIIth(facial), IXth (glossopharygeal), and Xth (vagus) cranial nerves. Untilmidgestation in the mouse, PHOX2b is expressed in the Vth (trigeminal)cranial nerve. In the mouse peripheral nervous system, PHOX2b isexpressed in the distal VIIth, IXth and Xth cranial sensory ganglia fromembryonic day 9.5 and in all autonomic nervous system ganglia as earlyas formed, until at least midgestation. Finally, by embryonic day 9-9.5,PHOX2b protein is detected in enteric neuroblasts invading the foregutmesenchyme; with expression in the esophagus, small intestine, and largeintestine. In the PHOX2b knock-out, the gut is devoid of enteric neuronsand even the neural crest-derived cells that are found in the foregut atE10.5 do not survive or migrate [Young et al., 1999]. Recognizing thephenotype of CCHS with symptoms of ANSD in the respiratory controlsystem (100% of CCHS probands), cardiovascular system (90% of CCHSprobands), opthalmologic system (88% of CCHS probands), neurologicalsystem (53% of probands), and gastrointestinal system (96% of probands)among subjects tested in the present examples, these findings followlogically from the embryologic distribution of PHOX2b. It remainsunclear how the distribution and actions of PHOX2b account forinvolvement of other systems often included in the ANSD profile of thechild with CCHS, including the sudomotor system (80% of CCHS probands),psychological system (30% of CCHS probands), and the renal system (29%of CCHS probands).

Polyalanine expansion mutations have been described as a cause ofdisease in a number of homeodomain- and non-homeodomain-containingtranscription factors including HOXD13 (synpolydactyly), HOXA13(hand-foot-genital syndrome), RUNX2 (cleidocranial dysplasia) and ZIC2(holoprosencephaly) [Goodman and Scambler, 2001]. There is precedent forpolyalanine repeat tract expansion in a homeobox gene as a cause ofneurological disease due to presumed failure of specification and/ormigration of a specific neuronal cell type. The Aristaless-relatedhomeobox gene (ARX) gene has been associated with XLAG (X-linkedlissencephaly and ambiguous genitalia) [Kitamura et al., 2002], X-linkedmental retardation [Bienvenu et al., 2002], X-linked and sporadicinfantile spasms, and other developmental disorders with mentalretardation and epilepsy [Stromme et al., 2002]. This ARX gene containsa polyalanine tract which is expanded in some affected subjects,particularly those with infantile spasms or myoclonic epilepsy. Othersubjects have missense or truncating mutations which likely result inloss-of-function. Mice with mutations in ARX show aberrantdifferentiation and migration of GABA-ergic neurons in neocortex[Kitamura et al., 2002]. Given the expression patterns of PHOX2b incentral autonomic structures and peripheral neural crest derivatives andthe wide range of ANS dysfunction seen in CCHS, there may be a similarmechanism of aberrant differentiation and/or migration of central andperipheral noradrenergic sympathetic and parasympathetic neurons resultsfrom the polyalanine tract expansion in PHOX2b. Nevertheless, one ofskill in the art will understand that other mechanisms may be importantin the ANS dysfunction seen in CCHS.

Without limiting the scope of the invention, the identification ofmutations in PHOX2b which cause typical CCHS and produce a verytruncated protein or a highly disrupted protein due to out-of-frameintragenic deletion suggests that the polyalanine expansion mutationresults in CCHS through a loss-of-function mechanism. However, nomutations which disrupt the homeodomain have thus far been identified,raising the possibility that if these very abnormal proteins are stable,they may still have some function. Indeed, polyalanine expansionmutations in HOXD13 and RUNX2 are thought to cause disease throughgain-of-function mechanisms. Polyalanine tracts are thought to act asspacers or protein-binding elements. Therefore, expansions of thesetracts in a mutant protein which can still bind DNA could prevent normalprotein interactions (loss-of-function) or allow aberrant interactions(gain-of-function) while blocking function of the normal protein codedby the non-mutated gene. This could explain the dominant inheritancepattern in a loss-of-function mutant. Further, in these models, longerrepeat tracts might be expected to produce a more pronounced moleculardisturbance, resulting in increasing severity and number of clinicalsymptoms with increased length of repeat tract, as observed in thepresent examples.

Clinically, evaluation of the PHOX2b expansion can be used as apredictive test for CCHS. Given that 65/67 cases and 0/67 controls inthe examples were heterozygous for the PHOX2b expansion, the sensitivityis ˜97.06%, with a specificity 100% when using individual embodiments ofthe methods and assays of the invention. Such tests could beparticularly useful for parents of a child with CCHS seeking recurrencerisk estimation or prenatal diagnosis, for grown children with CCHSseeking pre-conception or prenatal diagnosis, and for differentialdiagnosis for children with seemingly confounding symptoms such asasphyxia, prematurity, bronchopulmonary dysplasia, and more. Further, abetter understanding of the nature of the mutation may lead to improvedtreatment options for children with CCHS.

The results from the present examples are important at several levels.First, the study design serves as a paradigm of phenotypic featuresdirecting selection of candidate genes. Specifically, the symptoms ofANSD in the children with CCHS (and their relatives) motivated the studyof genes which play a role in early embryologic development of the ANS.This study design therefore has applicability to other seemingly complexdiseases that appear to be genetic in origin. Second, these resultsconfirm that children with CCHS are heterozygous for a PHOX2bpolyalanine expansion mutation. The identification of this mutation inmost individuals with CCHS allows a definitive confirmatory test forinfants suspected to have CCHS and allows for diagnostic homogeneity inCCHS cohorts used for research studies. Third, because the familystudies presented in the examples are consistent with an autosomaldominant pattern of inheritance for CCHS, certain embodiments of theinvention allow for careful informed pregnancy planning for families ofCCHS cases.

It will be understood to those skilled in the art that the presentinvention readily lends itself to automation. As an automated process,several samples containing the same, or different, combinations ofnucleic acids and/or proteins can be assayed in parallel.

The present invention also provides kits for carrying out the methodsdescribed herein. In one embodiment, the kit is made up of instructionsfor carrying out any of the methods described herein. The instructionscan be provided in any intelligible form through a tangible medium, suchas printed on paper, computer readable media, or the like. The presentkits can also include one or more reagents, buffers, hybridizationmedia, nucleic acids, primers, nucleotides, probes, molecular weightmarkers, enzymes, solid supports, databases, computer programs forcalculating dispensation orders and/or disposable lab equipment, such asmulti-well plates, in order to readily facilitate implementation of thepresent methods. Enzymes that can be included in the present kitsinclude nucleotide polymerases and the like. Solid supports can includebeads and the like whereas molecular weight markers can includeconjugatable markers, for example biotin and streptavidin or the like.Examples of preferred kit components can be found in the descriptionabove and in the following examples.

Some kits will include nucleic acid primers for amplifying the PHOX2bnucleic acids. Kits can also contain probes specific for the PHOX2b geneand gene products as described herein.

EXAMPLES

Study Subjects. Four distinct groups were investigated in this study,including CCHS probands, gender- and ethnicity matched controls, parentsand other relatives of CCHS probands, and CCHS parent/infant pairs. Forall subjects, ethnicity was assigned based on self-report. This studywas approved by the Rush University Medical Center institutional reviewboard and informed consent was obtained from all subjects and theirparents or legal guardians.

CCHS Cases. Sixty-nine CCHS probands (age range at enrollment 2 monthsto 22 years) with a diagnosis made by study in the RespiratoryPhysiology Laboratory at Rush Children's Hospital and/or followingthorough review of the medical records (DEW-M) were enrolled in thestudy. The diagnosis of CCHS was based on the accepted definition in theAmerican Thoracic Society Statement on the diagnosis and management ofIdiopathic CCHS. [Weese-Mayer et al., 1999]. Because DNA was notobtainable from 2 of the deceased children with CCHS, 67 CCHS probandswere included in the genetic studies.

Control Subjects. Sixty-seven unrelated control subjects were matchedfor ethnicity and gender to the CCHS cases with a 1:1 match ratio(collected at Rush Children's Hospital, Chicago, Ill.). After informedconsent was obtained, a three-generation family history was taken foreach control to ensure that no family member had a diagnosis of SIDS,Hirschsprung Disease, Idiopathic Congenital Central HypoventilationSyndrome, apparent life threatening event, primary (non-acquired)disorder of autonomic nervous system (ANS) dysregulation, or tumor ofneural crest origin.

Parents and Other Relatives of CCHS Cases. A total of 97 parents wereenrolled, including 43 sets of parents and 11 single parents (10mothers, 1 father), whose children were diagnosed with CCHS. Parentswere not available for 6 of the 7 adopted CCHS cases, or for the 4 CCHSmothers. A total of 30 siblings (20 sisters, 10 brothers), 6 half-sibs(2 half-sisters, 4 half-brothers), 7 grandparents (5 grandmothers, 2grandfathers), 1 aunt and 2 cousins were enrolled. Analyses on parentsand other relatives were limited to the PHOX2b gene polyalanine repeatmutation.

CCHS Cases with Offspring. A total of four parent/infant pairs in whichthe mother had CCHS were enrolled. Fathers for two of the infants werealso enrolled.

Subject Population Demographics. The matched sample dataset included 67CCHS probands and 67 matched controls with the following distribution ofcase-control pairs: 30 Caucasian females, 28 Caucasian males, 3African-American females, 2 African-American male pairs, 2 Hispanicfemales, 1 Hispanic male, and 1 Native American Indian female. Theparticipating parents of CCHS cases were all Caucasian with theexception of 3 African American parents (1 couple and 1 single mother),and 3 Hispanic parents (1 couple and 1 single mother). The CCHScase/offspring pairs included 1 Native American Indian (infant female),2 Caucasian (infant males), and 1 African American (infant male).

Among the children considered to have CCHS, 9 had unique diagnoses ofcerebral arteriovenous malformation [Mukhopadhyay and Wilkinson, 1990],cystic fibrosis, Tourette's, pervasive developmental delay (n=2),symptom manifestation after an acute infection (n=2), bronchopulmonarydysplasia, and brainstem hypoplasia. Among these 9 children, thesecondary diagnosis had clouded the presumptive diagnosis of CCHS.

DNA Preparation. Blood (3-10 cc) was obtained by venipuncture andcollected into an EDTA tube from CCHS cases and family members. Blood ora buccal swab was obtained from control subjects. Genomic DNA wasisolated utilizing a Puregene reagent kit (Gentra, Minneapolis, Minn.)according to the manufacturer's instructions. DNA samples were saved inTris-EDTA hydration buffer at −80° C. prior to genotyping. For two ofthe CCHS cases who had died several years earlier, paraffin-embeddedrectal biopsy specimens were obtained. Despite efforts to extract DNAfrom these specimens, this was not achieved.

Genotyping of PHOX2b Polyalanine Repeat Sequence. The PHOX2b exon 3region coding for the polyalanine repeat was amplified with primer pair5′-CCAGGTCCCAATCCCAAC-3′ (forward) (SEQ ID NO: 4) and5′-GAGCCCAGCCTTGTCCAG-3′ (reverse) (SEQ ID NO: 5) in a Perkin Elmer 9600thermal cycler (Applied Biosystems, Foster City, Calif.). The PCRreactions were carried out using 0.25 units AmpliTaq Gold polymerase(Applied Biosystems, Foster City, Calif.) in a total volume of 25 μlcontaining 50 ng genomic DNA, 0.3 μM primers, 2.5 mM MgCl₂, AmpliTaqGold PCR Buffer and 0.2 mM dNTPs with 70% 7-deazaGTP, 0.2uCi of (³²P)dCTP (Perkin Elmer/NEN Life Sciences, Boston, Mass.) and 10% glycerol.The amplification was performed with an initial denaturation at 95° C.for 10 min. followed by 35 cycles of denaturation at 94° C. for 30 sec.,annealing at 57° C. for 30 sec. and extension at 72° C. for 30 sec.Final extension was at 72° C. for 10 min. The PCR products (232 bp fornormal 20 repeat allele) were subjected to electrophoresis on a 6%denaturing polyacrylamide gel, and visualized by autoradiography. Allelerepeat number was determined by comparison of bands to known sizestandards for which repeat number had been determined by sequenceanalysis.

Sequence Analysis of PHOX2b. PHOX2b exons 1, 2 and 3 were amplified withprimer pairs [Garcia-Barceló et al., 2003] noted in Table I to giveproducts of 586 bp (exon 1), 442 bp (exon 2) and 687 bp (exon 3)utilizing a Perkin Elmer 9600 thermal cycler (Applied Biosystems, FosterCity, Calif.). The PCR reactions were carried out using 1.25 unitsAmpliTaq Gold polymerase (Applied Biosystems, Foster City, Calif.) in atotal volume of 25 μl containing 50 ng genomic DNA, 0.5 μM primers, 1 mMMgCl₂, AmpliTaq Gold PCR Buffer and 0.2 mM dNTPs. Due to its high GCcontent, amplification of exon 3 was performed using the GC-RICH System(Roche Molecular Biochemicals, Indianapolis, Ind.). Exon 3 PCR wasperformed in a volume of 25 μl containing 50 ng genomic DNA, 0.2 mMdNTPs, 5 ul of 5×GC-RICH PCR reaction buffer, 2.5 μl of 5M GC-RICHresolution solution, 0.5 uM of each primer, and 1.25 U of Taq DNAPolymerase mixture. The amplification for all 3 exons was performed withan initial denaturation at 95° C. for 8 min. followed by 35 cycles ofdenaturation at 95° C. for 1 min., annealing at 62° C. for 1 min., andextension at 72° C. for 45 sec. Final extension was at 72° C. for 10min. The PCR products were column-purified with a MinElute PCRPurification Kit (QIAGEN Inc., Valencia, Calif.) to remove reactionbuffer and unincorporated primers, and visualized by running 5 μl ofeach sample on 2% agarose gels. PCR products were sequenced on an ABI3100 automated sequencer (Research Resource Center, University ofIllinois at Chicago, Chicago, Ill.). The exon 3 reaction with theGC-Rich System was run using the primer pair flanking the polyalaninerepeat (provided in section above) and products were run on a 2% agarosegel for rapid screening for presence of a repeat expansion (FIG. 1).Polyacrylamide gel electrophoresis was necessary for accurate sizing ofmutations identified.

TABLE I 1 GACCTCAGACAAGGCATCTCA AATTACCCCTCCCTGCAATC (SEQ ID 6) (SEQ ID7) 2 CTGCCGTATGACCTGACCTT ACAGCCACACCAAATCCAGT (SEQ ID 8) (SEQ ID 9) 3ACCCTAACCGGTGCTTTTCT ACAATAGCCTTGGGCCTACC (SEQ ID 10) (SEQ ID 11)

Statistical Analysis. Association between the number of PHOX2bpolyalanine repeats and the quantitative traits (number of ANSD symptomsand number of affected systems as described in Weese-Mayer et al., 2001)was analyzed using a measured genotype approach [Boerwinkle et al.,1986; Boerwinkle et al., 1987]. Measured genotype analysis (MGA)consists of comparing the genotypic means by analysis of variance(ANOVA). Association between the number of polyalanine repeats and thequalitative trait “daily duration of required ventilatory support” wasassessed using λ2 contingency table tests. To address the issue of asparse contingency table, Monte Carlo tests of association asimplemented in computer algorithm CLUMP were used [Sham and Curtis,1995].

Results

Description of CCHS Matched Cohort. From the matched dataset, 12 of theCCHS cases had Hirschsprung Disease in addition to the 2 deceasedchildren from whom DNA was not obtainable (14 of 69 CCHS cases; 20%).Three of the CCHS cases had a tumor of neural crest origin (2 withganglioneuroma, 1 with neuroblastoma) in addition to one of the 2deceased children with CCHS who had a neuroblastoma (4 of 69 CCHS cases;5.8%). 54% of the CCHS cases required ventilatory support awake andasleep and 46% required ventilatory support during sleep only. Using thepublished list of ANSD symptoms in CCHS [Weese-Mayer et al., 2001], itwas determined that CCHS cases had a predominance of symptoms of ANSDwith a mean number of symptoms per subject of 9.17 (3.70) (range 3-20)and a mean number of systems involved per subject of 5.10 (1.32) (range2-8). Among the CCHS cases, system involvement included 90% withcardiovascular, 96% with gastrointestinal, 53% with neurological, 88%with opthalmologic, 29% with psychological, 17% with renal/urological,100% with respiratory, and 80% with sudomotor.

Directed Mutation Analysis of Polyalanine Repeat Sequence in PHOX2b inProbands with CCHS. A polyalanine repeat expansion mutation wasidentified in 65 of the 67 (97%) probands with CCHS for whom DNA wasavailable for analysis. All affected individuals with CCHS wereheterozygous for the mutation and carried a normal allele as well as theexpanded allele (FIG. 1). Expanded alleles contained insertions of 15-39nucleotides, increasing the normal polyalanine repeat present in PHOX2bfrom 20 alanines to 25-33 alanines (FIGS. 1, 2). Size of the expansionmutation was confirmed by direct sequencing for seven CCHS probandsamples with different repeat sizes; the inserted sequence wasidentified as additional alanine codons added in-frame to the 3′ end ofthe polyalanine coding tract. The majority of expanded alleles contained25-27 polyalanine repeats but some were larger (FIG. 2). No controlscarried an expanded sequence, although two out of 67 controls wereheterozygous for two deleted variants containing 15 repeats. Thisdeletion variant was identified previously in the control population byAmiel et al. [2003], in a study where two out of 125 controlsheterozygous for deleted variants containing 14 or 15 repeats.

PHOX2b Polyalanine Repeat Analysis in Parents and Other Relatives ofCCHS Cases. To determine if the repeat expansion mutation is inheritedand to clarify recurrence risk, 43 sets of parents and 11 single parentswhose children demonstrated the CCHS phenotype and for whom DNA wasavailable for analysis were studied. Most parents were homozygous forthe normal 20-repeat allele suggesting that most CCHS expansionmutations in the probands arose de novo. Four clinically unaffected (noCCHS) unrelated parents (representing ˜10% of families from which bothparents' DNA was available), however, did show an expansion mutationwhich was identical to that seen in the affected child. The expansionmutation in all four parents of probands showed a substantially lightersignal than that seen in the normal allele, in contrast to the patternseen in subjects with CCHS, in whom the normal allele and expandedallele had similar signal intensity (FIG. 1). This suggests that theseunaffected parents are most likely somatic mosaics for the expansionmutation. Three additional unrelated parents (of the 97 total) wereheterozygous for the deletion variant (2 with 15 repeats, 1 with 14repeats) seen in the normal population and thus, the frequency of thisvariant was essentially the same in parents of CCHS probands as in thegeneral population (allele frequency 0.015 in both control and parentgroups). One child with CCHS inherited the deletion variant from hisfather in addition to an expansion mutation, presumably from the mother(FIG. 1). All other relatives studied were homozygous for the normal20-repeat allele.

PHOX2b Polyalanine Repeat Analysis in CCHS Probands with Offspring. ThePHOX2b expansion mutation was present in all four CCHS female probands,who were mothers of one child each. Two of these infants had symptomscompatible with CCHS in early infancy, one did not demonstrate nocturnalventilatory dependence until after an acute respiratory infection, andone did not manifest any symptoms compatible with CCHS. The threesymptomatic children were heterozygous for an expansion mutationidentical in size to that seen in the mother (FIG. 3). The child with nosymptoms had a normal genotype and had inherited the mother's normalallele (FIG. 3). These mother-child pairs confirm the expected autosomaldominant inheritance pattern for individuals affected with CCHS andsuggest that repeat instability when the expansion mutation is passedfrom parent to child is not common. The two fathers who were testeddemonstrated the normal 20-repeat pattern.

PHOX2b Sequence Analysis in CCHS Probands with No Polyalanine ExpansionMutation. Two children with an established diagnosis of CCHS did notshow the expansion mutation. Sequence analysis of the full coding regionof PHOX2b including intron-exon boundaries identified a heterozygousnonsense mutation (A463T) in one of these probands diagnosed with CCHS,Hirschsprung disease, and neuroblastoma. This mutation introduces a stopcodon at K155 and is expected to produce a truncated protein which ismissing most of exon 3 and the entire polyalanine tract. The otherproband showed a single base change in exon 3 (G875C) which resulted ina substitution of alanine for glycine (G292A); this change was alsofound in one control subject. Thus, this one CCHS proband did not showany significant abnormality in PHOX2b. However, he had an atypicalclinical picture with reported brainstem hypoplasia and severe mentalretardation in addition to his very characteristic picture of ANSD;further, he had a previously reported EDN3 frameshift point mutation[Bolk et al., 1996].

Relationship of Polyalanine Repeats to ANSD Phenotype in CCHS Cases.Measured genotype analysis revealed a significant association betweenPHOX2b polyalanine repeat mutation length and number of ANSD symptoms(F=2.93, df=5, p=0.021; FIG. 4), but not number of ANSD-affected systemsinvolved (F=1.80, df=5, p=0.129). There was also a significantassociation between the distribution of repeat mutation length and dailyduration of required ventilatory support (Ti=17.667, p=0.0034; TableII).

TABLE II Frequency Distribution of PHOX2b Polyalanine Repeats by DailyDuration of Ventilatory Support* Number of PHOX2b Polyalanine Repeats**25 26 27 28 30 33 24 hours 0 16 14 0 2 1 12 hours 8 7 8 1 0 4 *Becausethe daily duration of ventilatory support was unknown for 4 CCHSprobands, and 2 probands did not demonstrate the PHOX2b polyalanineexpansion mutation, the total number of subjects in this table is 61.Also, decisions about daily duration of ventilatory support were made bythe referring pulmonologist. In the four cases with 12 hour/day supportand the 13 additional repeats (33 total polyalanine repeats), review ofthe medical records (performed independent of the genetic testingresults) indicates markedly elevated awake carbon dioxide values and anabsence of any current awake physiologic evaluations; by Rush laboratorycriteria review of records, 24 hour/day ventilatory support would havebeen recommended to 3 of these 4 children with the 13 additionalrepeats. **P = 0.0034 for difference in allele distribution.

The present compositions can have any or all of the components describedherein, including but not limited to primers, samples, and the like.Likewise, the present methods can be carried out by performing any ofthe steps described herein, either alone or in various combinations. Oneskilled in the art will recognize that all embodiments of the presentinvention are capable of use with all other appropriate embodiments ofthe invention described herein. Additionally, one skilled in the artwill realize that the present invention also encompasses variations ofthe present probes, configurations and methods that specifically excludeone or more of the components or steps described herein.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” “more than”and the like include the number recited and refer to ranges which can besubsequently broken down into subranges as discussed above. In the samemanner, all ratios disclosed herein also include all subratios fallingwithin the broader ratio.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, thepresent invention encompasses not only the entire group listed as awhole, but each member of the group individually and all possiblesubgroups of the main group. Accordingly, for all purposes, the presentinvention encompasses not only the main group, but also the main groupabsent one or more of the group members. The present invention alsoenvisages the explicit exclusion of one or more of any of the groupmembers in the claimed invention.

All references, patents and publications disclosed herein arespecifically incorporated by reference thereto. Unless otherwisespecified, “a” or “an” means “one or more”.

The following references, some of which are discussed herein, arespecifically incorporated into the present application by reference:

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While preferred embodiments have been illustrated and described, itshould be understood that changes and modifications can be made thereinin accordance with ordinary skill in the art without departing from theinvention in its broader aspects as defined in the following claims.

1. A primer set for amplifying the second polyalanine repeat of a PHOX2bgene or gene product of a subject to diagnose or confirm idiopathiccongenital central hypoventilation syndrome (CCHS), comprising: (a) aforward primer selected from the group consisting of SEQ ID 4, SEQ ID 6,SEQ ID 8, and SEQ ID 10; and (b) a reverse primer selected from thegroup consisting of SEQ ID 5, SEQ ID 7, SEQ ID 9, and SEQ ID
 11. 2. Theprimer set of claim 1 wherein the primer set amplifies an expansionmutation in a PHOX2b gene or gene product.
 3. A kit for performing anamplification of a polyalanine repeat in a PHOX2b gene or gene productcomprising: instructions for performing amplification; and primerscapable of amplifying a polyalanine repeat in a PHOX2b gene or geneproduct.
 4. A fragment of the PHOX2b gene that is amplified by the setof primers designated by SEQ ID NOS: 4 and
 5. 5. Polymerase chainreaction products resulting from amplification of the PHOX2b gene byprimers designated by SEQ ID NO: 4 and 5 or functional equivalentsthereof.
 6. A forward and a reverse primer designated by SEQ ID NOS: 4and 5 or functional equivalents thereof.